The recent discoveries that opioid receptors, which are members of the G-protein coupled receptor (GPCR) family, can exist as heteromers in live cells, and that 8-|x and 6-K opioid receptor complexes are distinct functional signaling units, have added a new dimension of complexity to the opioid research field. Structural and mechanistic information about opioid receptor dimeric/oligomeric complexes has therefore become of major importance for understanding the mechanisms of action of opiates. The goal of the work proposed in this grant application is to identify the molecular determinants responsible for the oligomerization of 8- and |>opioid receptors (both homo- and heteromers) in a structural context of receptor models, using an iterative combined computational and experimental approach. Since there is no detailed structural data about opioid receptor dimers, and no evidence that the homo- and hetero-oligomerization interfaces of GPCRs coincide, we developed two different computational approaches based on correlated mutation analysis, and identified the most likely homo- and hetero- dimerization interfaces between the transmembrane regions of opioid receptor subtypes. Inferences from these bioinformatics tools and initial molecular models obtained in the preliminary studies constitute the pilot data for the combined computational and experimental strategy that will serve, as described in this grant application, to probe, validate, and refine models of 8- and |i-opioid receptor homo- and heteromers. An important goal of these studies is to be able to identify specific mutations that can be used to modulate dimerization/oligomerization, and thus affect receptor function in a manner that will reveal physiologically relevant mechanisms that depend and/or ensue from dimerization. The combined computational and experimental strategy proposed in this grant application is also expected to provide direct evidence either for the formation of subtype-specific interface(s) in 8- and |i- opioid receptor dimers, or for the arrangement of these receptors into higher-order oligomers with distinct symmetrical or asymmetrical interfaces, as recently proposed for rhodopsin oligomers. Inferences from these studies are expected to provide new insights into the mechanisms underlying opioid-receptor function, with the ultimate goal of helping drug design.